1. Field of the Invention
The present invention relates to methods and materials for assaying mammalian blood and tissue. More specifically it relates to methods and materials for determining the amounts of vitamin B.sub.12 and vitamin B.sub.12 analogues in human plasma.
2. Prior Art
For many years it has been recognized that the assay of the vitamin B.sub.12 level in humans is a valuable technique for diagnosing and subsequently treating certain diseases, such as for example, pernicious anaemia, post gastrectomy states nutritional deficiencies, intestinal disorders, and others. Initially, vitamin B.sub.12 was assayed microbiologically using either Euglena gracilis or Lactobacillus leichmannii. More recently, radioisotope dilution (RID) assays for B.sub.12 have been utilized. Such radioisotope dilution assay techniques are well documented in the literature, see for example Lau, et.al. (1965) "Measurement of Serum B.sub.12 Levels Using Radioisotope Dilution and Coated Charcoal," BLOOD, 26, 202, as modified by Raven et.al. (1968) "The Effect of Cyanide Serum and Other Factors on the Assay of Vitamin B.sub.12 by Radio-Isotope Method Using .sup.57 Co-B.sub.12, Intrinsic Factor and Coated Charcoal," GUYS HOSPITAL REPORTS, 117, 89; and (1969) "Improved Method for Measuring Vitamin B.sub.12 in Serum Using Intrinsic Factor, .sup.57 Co-B.sub.12 and Coated Charcoal," JOURNAL OF CLINICAL PATHOLOGY, 22, 205.
Such prior art radioisotope dilution assay of vitamin B.sub.12 generally includes the steps of freeing the endogenous B.sub.12 from its natural binding protein by boiling at a selected pH and then adding a measured amount of the radioisotope .sup.57 Co-B.sub.12 and a limited amount of binding protein. All of the binding protein will be bound by some form of B.sub.12 since the amount of radioisotope B.sub.12 added is, by itself, sufficient to bind the small amount of protein. As both the natural B.sub.12 and the radioactive B.sub.12 compete to bind with the protein, the degree to which the radioactive count of the protein bound B.sub.12 was inhibited was thought to be indicative of the amount of natural B.sub.12 present in the sample undergoing testing.
More specifically, in the technique of Lau et.al. as modified by Raven et.al., serum B.sub.12 is separated from binding protein in the plasma sample by boiling with 0.25 N HCl. Radioisotope B.sub.12 is added to the reaction mixture and the B.sub.12 mixture is reacted with protein, normally in the form of a commercially available binder. Then the free or unbound B.sub.12 is separated from the protein bound B.sub.12 by protein-coated charcoal and the radioactivity of the supernatant liquid containing the mixture of bound radioactive B.sub.12 and bound non-radioactive B.sub.12 counted for radioactivity. The serum B.sub.12 concentration is then calculated from the count, often by comparison with a standard chart. Almost as soon as this technique began to be utilized it was recognized that the vitamin B.sub.12 measurements it provided were usually inconsistent with the results obtained by other measuring techniques for B.sub.12, such as the microbiological assays. Most often, the vitamin B.sub.12 assay obtained by radioisotope dilute techniques have been found to be high. Many theories have been advanced to explain the cause of the high vitamin B.sub.12 readings. However, it is believed that nowhere in the prior art is it recognized that there are substances in mammalian blood and tissue which react with certain non-specific protein binders in the radioisotope dilution assay techniques to provide an analysis of vitamin B.sub.12 which is apparently higher than the amount of B.sub.12 actually in the sample. Additionally, it is believed that nowhere in the prior art is it recognized that most common and commercial RID assay protein binders are not specific to vitamin B.sub.12, but that they are also capable of binding with the heretofore unknown B.sub.12 analogues and thus provide erroneous B.sub.12 assays.